Alcohol and HIV Decrease Proteasome and Immunoproteasome Function in Macrophages: Implications for Impaired Immune Function During Disease

Proteasomes (proteinase complexes, PR) and immunoproteasomes (IPR) degrade damaged proteins and affect protein processing required for antigen presentation by mononuclear phagocytes. These critical immune processes are attenuated during progressive HIV-1 infection and are affected by alcohol abuse. To investigate the mechanisms underlying these functional changes, we measured PR and CYP2E1 activities [an ethanol (EtOH) metabolizing enzyme] and reactive oxygen species (ROS) in human monocyte-derived macrophages (MDM) following HIV-1 infection and EtOH treatment. We observed progressive declines of PR activity and PR/IPR contents in HIV-1-infected MDM. PR activity and IPR expression increased after IFN-γ stimulation but reduced after HIV-1 infection. EtOH inhibited both IFN-γ -induced PR and IPR. Paradoxically, EtOH attenuated PR catalytic activity in infected MDM and suppressed viral replication. Elevated ROS followed EtOH exposure and paralleled decreased PR activity. The latter was restored by anti-oxidant. The data support the notion that HIV-1 infection and EtOH may work in concert to affect immune function including antigen presentation and thereby affect disease progression

Soybean-derived Bowman-Birk inhibitor inhibits neurotoxicity of LPS-activated macrophages

<p>Abstract</p> <p>Background</p> <p>Lipopolysaccharide (LPS), the major component of the outer membrane of gram-negative bacteria, can activate immune cells including macrophages. Activation of macrophages in the central nervous system (CNS) contributes to neuronal injury. Bowman-Birk inhibitor (BBI), a soybean-derived protease inhibitor, has anti-inflammatory properties. In this study, we examined whether BBI has the ability to inhibit LPS-mediated macrophage activation, reducing the release of pro-inflammatory cytokines and subsequent neurotoxicity in primary cortical neural cultures.</p> <p>Methods</p> <p>Mixed cortical neural cultures from rat were used as target cells for testing neurotoxicity induced by LPS-treated macrophage supernatant. Neuronal survival was measured using a cell-based ELISA method for expression of the neuronal marker MAP-2. Intracellular reactive oxygen species (ROS) production in macrophages was measured via 2', 7'-dichlorofluorescin diacetate (DCFH₂DA) oxidation. Cytokine expression was determined by quantitative real-time PCR.</p> <p>Results</p> <p>LPS treatment of macrophages induced expression of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) and of ROS. In contrast, BBI pretreatment (1-100 μg/ml) of macrophages significantly inhibited LPS-mediated induction of these cytokines and ROS. Further, supernatant from BBI-pretreated and LPS-activated macrophage cultures was found to be less cytotoxic to neurons than that from non-BBI-pretreated and LPS-activated macrophage cultures. BBI, when directly added to the neuronal cultures (1-100 μg/ml), had no protective effect on neurons with or without LPS-activated macrophage supernatant treatment. In addition, BBI (100 μg/ml) had no effect on N-methyl-D-aspartic acid (NMDA)-mediated neurotoxicity.</p> <p>Conclusions</p> <p>These findings demonstrate that BBI, through its anti-inflammatory properties, protects neurons from neurotoxicity mediated by activated macrophages.</p

CB2 Receptor Activation Inhibits Melanoma Cell Transmigration through the Blood-Brain Barrier

During parenchymal brain metastasis formation tumor cells need to migrate through cerebral endothelial cells, which form the morphological basis of the blood-brain barrier (BBB). The mechanisms of extravasation of tumor cells are highly uncharacterized, but in some aspects recapitulate the diapedesis of leukocytes. Extravasation of leukocytes through the BBB is decreased by the activation of type 2 cannabinoid receptors (CB2); therefore, in the present study we sought to investigate the role of CB2 receptors in the interaction of melanoma cells with the brain endothelium. First, we identified the presence of CB1, CB2(A), GPR18 (transcriptional variant 1) and GPR55 receptors in brain endothelial cells, while melanoma cells expressed CB1, CB2(A), GPR18 (transcriptional variants 1 and 2), GPR55 and GPR119. We observed that activation of CB2 receptors with JWH-133 reduced the adhesion of melanoma cells to the layer of brain endothelial cells. JWH-133 decreased the transendothelial migration rate of melanoma cells as well. Our results suggest that changes induced in endothelial cells are critical in the mediation of the effect of CB2 agonists. Our data identify CB2 as a potential target in reducing the number of brain metastastes originating from melanoma

Anti-Inflammatory Effect of Targeted Delivery of SOD to Endothelium: Mechanism, Synergism with NO Donors and Protective Effects In Vitro and In Vivo

Pro-inflammatory activation of vascular endothelium is implicated in pathogenesis of severe conditions including stroke, infarction and sepsis. We have recently reported that superoxide dismutase (SOD) conjugated with antibodies (Ab/SOD) that provide targeted delivery into endothelial endosomes mitigates inflammatory endothelial activation by cytokines and agonists of Toll-like receptors (TLR). The goal of this study was to appraise potential utility and define the mechanism of this effect. Ab/SOD, but not non-targeted SOD injected in mice alleviated endotoxin-induced leukocyte adhesion in the cerebral vasculature and protected brain from ischemia-reperfusion injury. Transfection of endothelial cells with SOD, but not catalase inhibited NFκB signaling and expression of Vascular Cell Adhesion Molecule-1 induced by both cytokines and TLR agonists. These results affirmed that Ab/SOD-quenched superoxide anion produced by endothelial cells in response to proinflammatory agents mediates NFκB activation. Furthermore, Ab/SOD potentiates anti-inflammatory effect of NO donors in endothelial cells in vitro, as well as in the endotoxin-challenged mice. These results demonstrate the central role of intracellular superoxide as a mediator of pro-inflammatory activation of endothelium and support the notion of utility of targeted interception of this signaling pathway for management of acute vascular inflammation

PARP inhibition protects against alcoholic and non-alcoholic steatohepatitis

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Phosphorylation of Claudin-5 and Occludin by Rho Kinase in Brain Endothelial Cells

Critical to the proper maintenance of blood-brain-barrier (BBB) integrity are the endothelial tight junctions (TJs). Posttranslational modifications of essential endothelial TJ proteins, occludin and claudin-5, contribute and possibly disrupt BBB integrity. Our previous work has shown that Rho kinase (RhoK) activation mediates occludin and claudin-5 phosphorylation resulting in diminished barrier tightness and enhanced monocyte migration across BBB in the setting of human immunodeficiency virus-1 encephalitis (HIVE). To determine whether RhoK can directly phosphorylate TJ proteins, we examined phosphorylation of cytoplasmic domains of recombinant claudin-5 and occludin by RhoK. We found that RhoK predominately phosphorylated two sites on occludin (T382 and S507) and one site on claudin-5 (T207). Specific anti-phosphopeptide antibodies were developed for these sites, allowing the detection of phosphorylated occludin at T382 and S507, and claudin-5 at T207 from full-length recombinant occludin and claudin-5 transiently expressed in COS-7 cells and mouse brain microvascular endothelial cells. Finally, these phosphospecific antibodies demonstrated enhanced staining of brain endothelial cells in the mouse model for HIVE and human HIVE brains featuring mononuclear cell infiltration across disrupted BBB. Our results demonstrated the direct phosphorylation of occludin and claudin-5 by RhoK at specific sites, which was increased in encephalitic brain tissue. These antibodies could be useful reagents for monitoring BBB dysfunction in vivo

Blocking of P2X7r Reduces Mitochondrial Stress Induced by Alcohol and Electronic Cigarette Exposure in Brain Microvascular Endothelial Cells

Studies in both humans and animal models demonstrated that chronic alcohol/e-cigarette (e-Cig) exposure affects mitochondrial function and impairs barrier function in brain microvascular endothelial cells (BMVECs). Identification of the signaling pathways by which chronic alcohol/e-Cig exposure induces mitochondrial damage in BMVEC is vital for protection of the blood–brain barrier (BBB). To address the issue, we treated human BMVEC [hBMVECs (D3 cell-line)] with ethanol (ETH) [100 mM], acetaldehyde (ALD) [100 μM], or e-cigarette (e-Cig) [35 ng/mL of 1.8% or 0% nicotine] conditioned medium and showed reduced mitochondrial oxidative phosphorylation (OXPHOS) measured by a Seahorse analyzer. Seahorse data were further complemented with the expression of mitochondrial OXPHOS proteins detected by Western blots. We also observed cytosolic escape of ATP and its extracellular release due to the disruption of mitochondrial membrane potential caused by ETH, ALD, or 1.8% e-Cig exposure. Moreover ETH, ALD, or 1.8% e-Cig treatment resulted in elevated purinergic P2X7r and TRPV1 channel gene expression, measured using qPCR. We also demonstrated the protective role of P2X7r antagonist A804598 (10 μM) in restoring mitochondrial oxidative phosphorylation levels and preventing extracellular ATP release. In a BBB functional assay using trans-endothelial electrical resistance, we showed that blocking the P2X7r channel enhanced barrier function. In summary, we identified the potential common pathways of mitochondrial injury caused by ETH, ALD, and 1.8% e-Cig which allow new protective interventions. We are further investigating the potential link between P2X7 regulatory pathways and mitochondrial health